Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC)

Abstract

The conformational conversion of the normal cellular prion protein (PrPC) into the pathology-associated PrPSc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPC molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPC, PrPSc is insoluble in non-ionic detergents and does not bind to Cu2+ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-beta-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu2+-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrPC and denatured PrPSc were retained by a Cu2+-loaded resin, native PrPSc and PrPres [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPC from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrPC from aggregated PrPSc in infected brains. Our results indicate that in contrast with PrPSc in uninfected as well as infected brains, PrPC is predominantly present in the glycosylated forms.

Figure 1. Subcellular localization of PrP

PrP^C enriched in cell fractions of uninfected bovine stem brain (A), and PrP^C/PrP^Sc enriched in cell fractions of BSE-infected bovine stem brain (B). Brain samples were homogenized, subcellular fractionated and immunoblotted as described. PrP contents of cytoplasmic membrane fraction (lane 1), mitochondrial fraction (lane 2), synaptosomal/microsomal fraction (lane 3), sucrose phase (lane 4), supernatant of the ultracentrifugation (lane 5) and a positive control comprising 10 ng of recbPrP (lane 6) were determined using a mix of the anti-PrP mabs 3B5, 4F2, 8G8, 11C6, 12F10 and 14D3. The diglycosylated (a), monoglycosylated (b) and unglycosylated (c) PrP forms are indicated. Molecular masses are indicated in kDa.

Figure 2. Partial purification of PrP^C by IMAC

Octylglucoside extracts from normal mouse brain (A) and normal bovine stem brain (B) were applied to and eluted from a Cu²⁺-loaded IMAC column. Flow-through samples (A, lanes 1–8, total protein in each lane, 90 μg; B, lanes 1–2, total protein in each lane, 35 μg), a positive control comprising recbPrP (A, lane 9; B, lane 6; 10 ng of each), washes (A, lanes 10–12, total protein in each lane, 90 μg; B, lanes 3–5, total protein in each lane, 35 μg) and eluted samples (A, lanes 13–15, total protein in each lane, 15 μg; B, lanes 7–10, total protein in each lane, 55 μg) were immunoblotted using a mixture of the anti-PrP mabs 3B5, 4F2, 8G8, 11C6, 12F10 and 14D3. PrP^C was detected in the eluated samples. Molecular masses are indicated in kDa.

Figure 3. PrP^C and PrP^Sc glycotyping patterns of murine specimens after IMAC

Western blot signals after IMAC were quantified with image analysis software. For this to be carried out, all PrP-specific signals in the flow-through were regarded as being PrP^Sc, and all PrP-specific signals in the eluate were regarded as PrP^c. Mean contributions (%) of di-, mono- and un-glycosylated PrP bands to the combined signals (set as 100%) are shown. The numbers (n) of specimens examined are given.

Figure 4. Partial purification and separation of PrP^C and PrP^Sc by IMAC

Octylglucoside extracts from mouse brain infected with the scrapie strain Chandler (A) and BSE-infected bovine stem brain (B) were applied to and eluted from a Cu²⁺-loaded IMAC column. Flow-through samples (lanes 1–4, total protein in each lane, 15 μg), a positive control comprising recbPrP (lane 5, 10 ng), washes (lanes 6–8, total protein in each lane, 15 μg) and eluted samples (lanes 9–12, total protein in each lane, 50 μg) were immunoblotted as described in Figure 1. The diglycosylated (a), monoglycosylated (b) and unglycosylated (c) PrP forms in the eluted samples are indicated. Molecular masses are indicated in kDa.

Figure 5. PK digestion of IMAC fractions of scrapie-infected murine brain and BSE-infected bovine stem brain

Octylglucoside extracts from murine brain infected with the scrapie strain Chandler (A) and BSE-infected bovine stem brain (B) were applied to and eluted from a Cu²⁺-loaded IMAC column. Pooled flow-through samples (500 μg/ml total protein), pooled eluted samples (1000 μg/ml total protein) and solubilized samples immediately prior to application to the Cu²⁺-loaded column (750 μg/ml total protein) were digested for 30 min at 37 °C in the presence of the PK concentrations indicated. In case of the flow-through sample in (A), digested with 0.5 μg/ml PK, 750 μg/ml total protein was used. After SDS/PAGE (total protein in each lane, 30–50 μg) immunoblotting was carried as described in Figure 1. The positive controls (Pos) comprised 15 ng of recbPrP. Molecular masses are indicated in kDa.

Figure 6. Partial purification of denatured PrP isoforms by IMAC

Octylglucoside extracts from murine brain infected with the scrapie strain Chandler were denatured by boiling in 4% SDS for 10 min and applied to and eluted from a Cu²⁺-loaded IMAC resin (A). PrP-containing eluate fractions were pooled and digested for 30 min at 37 °C in the presence of PK at the concentrations indicated (B). Flow-through samples (A, lanes 1–4, total protein in each lane, 25 μg), a positive control comprising recbPrP (A, lane 5; B, Pos; 10 ng of each sample), washes (A, lanes 6–8, 25 μg total protein each), eluted samples (A, lanes 9–12, total protein in each lane, 25 μg) and PK-digested pooled eluate fractions (B, 500 μg/ml each) were immunoblotted as described in Figure 1. The diglycosylated (a), monoglycosylated (b) and unglycosylated (b) PrP forms in the eluted samples are indicated. Molecular masses are indicated in kDa.

Figure 7. Partial purification of denatured and PK digested PrP isoforms by IMAC

Octylglucoside extracts from murine brain infected with the scrapie strain Chandler (lane 1, 15 μg total protein) were digested for 30 min at 37 °C in the presence of 1 μg/ml PK (lane 2, total protein, 15 μg) and denatured by boiling in 10% SDS for 10 min. Subsequently the sample was applied to and eluted from a Cu²⁺-loaded IMAC column and flow-through samples (lanes 3–4, total protein in each lane, 10 μg), washes (lanes 5–7, total protein in each lane, 19 μg) and eluted samples (lanes 8–11, total protein in each lane, 30 μg) were immunoblotted as described in Figure 1. Molecular masses are indicated in kDa.